A simple test procedure is described for the rapid evaluation of β-lactamase substrate profiles against all the clinically important β-lactam antibiotics. By use of this method 100 clinical isolates of Escherichia coli were classified into four β-lactamase types: TEM-like, 35; chromosomal-like, 56; others, 3; β-lactamase-less, 6 strains. They were tested for their susceptibilities to cefuroxime, cefoxitin, cefamandole, cephalexin, cefazolin, cephaloridine, cephalothin, ampicillin, carbenicillin and kanamycin. The isolates of each of TEM-like and chromosomal-like β-lactamase types showed a good correlation between the β-lactamase activity and the minimum inhibitory concentration (MIC) determined by the agar dilution method and the least square line analysis; MIC = ax + b, where x is enzyme activity, and a and b are constants. TEM-like β-lactamase increased the resistance to ampicillin and carbenicillin markedly and that to cephalothin, cephaloridine and cefamandole moderately. In contrast, chromosomal-like β-lactamase increased the resistance to ampicillin, cephalexin and cephalothin only s